1. Project summary: The long-range goal of the project is to understand the mechanism of cytokinesis in enough detail to make useful mathematical models of the process that can predict the results of future experiments. Our experimental organism, the fission yeast Schizosaccharomyces pombe, has been highly advantageous for this work. Over the past four years we made progress toward this goal by determining the structure of cytokinetic nodes, the organizing centers for the contractile ring, by super resolution light microscopy, showing that actin filaments shorten as the contractile ring constricts, finding that the Septation Initiation Network (SIN) controls the assembly of type I interphase nodes, but ruling out SIN as the trigger for contractile ring constriction and discovering that at least two pathways, recruitment of -glucan synthase Bgs1p and nucleation of microtubules from the contractile ring, contribute to trigger ring constriction. In spite of this progress, essential details are still missing and will be addressed by four lines of research. Our first goal is to determine the molecular organization of the fission yeast contractile ring. We will use quantitative super resolution fluorescence microscopy to make a complete, quantitative inventory of fission yeast cytokinesis proteins and the structures that they form from interphase through the end of mitosis. The second goal is to measure protein turnover in cytokinesis nodes and contractile rings. Our computer simulations of contractile ring constriction revealed that turnover of actin filaments, formins and myosin-II is required to produce tension. The most likely mechanism of exchange is association and dissociation of individual protein molecules over time, but our super resolution fluorescence microscopy data indicate that whole nodes may appear and disappear by a mysterious mechanism during cytokinesis. We will use a new microscopy method to measure the exchange of node and contractile ring proteins with cytoplasmic pools with sufficient spatial resolution to distinguish the exchange of single molecules and whole structures. The third goal is to model contractile ring assembly and constriction. We will use the data from the first two projects to update the mathematical models and simulations of contractile ring assembly in collaboration with Dimitrios Vavylonis and of tension generation during constriction in collaboration with Ben O?Shaughnessy. The fourth project is to characterize how -glucan synthase Bgs1p and microtubules nucleated from the contractile ring function redundantly to trigger ring constriction. These projects are powered by innovative methods to count protein molecules and measure their turnover by high speed FPALM super resolution microscopy. Given the evolutionary conservation of many of the participating molecules, I believe that studies of fission yeast will establish the basic molecular pathways controlling cytokinesis in other eukaryotes. 1